RESUMEN
Adhesive interactions between molecules on tumor cells and those on target organs play a key role in organ specific metastasis. Poly-N-acetyl-lactosamine (polyLacNAc) substituted N-oligosaccharides on melanoma cell surface glycoproteins promote lung specific metastasis via galectin-3 by facilitating their arrest and extravasation. This study reports the identification and characterization of galectin-3 interacting proteins using a combination of galectin-3 sepharose affinity and leucoagglutinating phytohemagglutinin (L-PHA) columns. A total of 83 proteins were identified as galectin-3 interacting glycoproteins, of which 35 were constituents of the L-PHA bound fraction, suggesting that these proteins carry polyLacNAc substituted ß1,6 branched N-glycans. The identities of some of these proteins, like LAMP-1, LAMP-3, basigin, embigin, and α5 and ß1 Integrin, have been confirmed by western blotting, and functional relevance with respect to metastatic properties has been established.
Asunto(s)
Proteínas Portadoras/metabolismo , Galectina 3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Espectrometría de Masas , Melanoma/patología , Mapeo de Interacción de Proteínas/métodos , Animales , Cromatografía de Afinidad , Cromatografía Liquida , Espectrometría de Masas/métodos , Melanoma Experimental , Ratones , Unión Proteica , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
Breast cancer, especially ER positive/HER2/neu negative IDC, is the predominant subtype of invasive ductal carcinoma. Although proteomic approaches have been used towards biomarker discovery in clinical breast cancer, ER positive/HER2/neu negative IDC is the least studied subtype. To discover biomarkers, as well as to understand the molecular events associated with disease progression of estrogen receptor positive/HER2/neu negative subtype of invasive ductal carcinoma, differential protein expression profiling was performed by using LC-MS(E) (MS at elevated energy). A total of 118 proteins were identified, of which 26 were differentially expressed. These identified proteins were functionally classified and their interactions and coexpression were analyzed by using bioinformatic tools PANTHER (Protein Analysis THrough Evolutionary Relationships) and STRING (Search Tool for the Retrieval of Interacting Genes). These proteins were found to be upregulated and were involved in cytoskeletal organization, calcium binding, and stress response. Interactions of annexin A5, actin, S100 A10, glyceraldehyde 3 phosphate dehydrogenase, superoxide dismutase 1, apolipoprotein, fibrinogen, and heat shock proteins were prominent. Differential expression of these proteins was validated by two-dimensional gel electrophoresis and Western blot analysis. The cluster of these proteins may serve as a signature profile for estrogen receptor positive/ HER2/neu negative subtype.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Ductal/metabolismo , Proteómica , Receptores de Estrógenos/metabolismo , Secuencia de Aminoácidos , Western Blotting , Neoplasias de la Mama/patología , Carcinoma Ductal/patología , Femenino , Genes erbB-2 , Humanos , Datos de Secuencia Molecular , Invasividad Neoplásica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Proteomic approaches aid in gaining a better understanding of the pathophysiology of diabetic complications. In view of this, differential protein expression in diabetic plasma samples was studied by a combination of proteomic and western blot analyses. Diabetic plasma samples were categorized based on glycated haemoglobin levels as controlled diabetes (CD; 7-8%), poorly controlled diabetes (PCD; >8%) and non-diabetic control (ND;<6.4%). Two-dimensional electrophoresis and liquid chromatographymass spectrometry revealed differential expression of proteins including upregulation of fibrinogen and haptoglobin and downregulation of vitamin D binding protein, α-1-antitrypsin, transthyretin and apolipoprotein A1 (Apo A1) in diabetic compared with non-diabetic plasma samples. Amongst these proteins, Apo A1 downregulation was prominent in PCD. Downregulation of Apo A1 may serve as an early predictive marker of diabetic complications.
Asunto(s)
Apolipoproteína A-I/sangre , Cromatografía Líquida de Alta Presión , Diabetes Mellitus/metabolismo , Espectrometría de Masas , Proteoma/análisis , Diabetes Mellitus/patología , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Hemoglobina Glucada/análisis , Humanos , Proyectos PilotoRESUMEN
Glycation, a nonenzymatic reaction between reducing sugars and proteins, is a proteome wide phenomenon, predominantly observed in diabetes due to hyperglycemia. Glycated proteome of plasma, kidney, lens, and brain are implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative diseases, cancer, and aging. This review discusses the strategies to characterize protein glycation, its functional implications in different diseases, and intervention strategies to protect the deleterious effects of protein glycation.
Asunto(s)
Glicosilación , Proteoma/metabolismo , Proteómica , Animales , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Proteoma/químicaRESUMEN
Cancer is associated with increased glycolysis and carbonyl stress. In view of this, AGE modified proteins were identified from clinical breast cancer tissue using 2DE-immunoblot and mass-spectrometry. These proteins were identified to be serotransferrin, fibrinogen gamma chain, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, annexin II, prohibitin and peroxiredoxin 6, which have established role in cancer. Further, RAGE expression and its downstream signaling proteins NADPH oxidase and NF-kB were studied. Role of these AGE modified proteins and RAGE signaling in breast cancer is discussed.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Productos Finales de Glicación Avanzada/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas de Neoplasias/metabolismo , Receptor para Productos Finales de Glicación Avanzada/biosíntesis , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Invasividad Neoplásica , Procesamiento Proteico-Postraduccional , Receptor ErbB-2/análisis , Receptor ErbB-2/metabolismoRESUMEN
Post-translational modifications (PTMs) are very important to biological function, however their identification and characterization is technically challenging. In this study, we have identified glycation modifications by nano LC-MSE, a data independent acquisition work flow, followed by database search using the Protein Lynx Global Server (PLGSJ). PLGS search with a complete human protein database hardly identified glycation modifications in a glycated human serum albumin (HSA), which was detected to be glycated by western blotting with advanced glycation end products (AGE) antibody and fluorescence spectroscopy. To overcome this difficulty, "Zoom-In" approach, a targeted database search was used to identify glycation modifications in a glycated HSA, which were further manually validated. This approach was useful for identification of glycation modifications from untargeted tandem mass spectrometryworkflow such as MSE, but may require the development of a new algorithm or an upgrade of the existing software.
Asunto(s)
Bases de Datos de Proteínas , Productos Finales de Glicación Avanzada/análisis , Proteínas/química , Albúmina Sérica/química , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Western Blotting , Glicopéptidos/química , Glicopéptidos/metabolismo , Glicosilación , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Albúmina Sérica/metabolismo , Espectrometría de FluorescenciaRESUMEN
Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.
Asunto(s)
Diabetes Mellitus/sangre , Hemoglobina Glucada/metabolismo , Glicoproteínas/sangre , Albúmina Sérica/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Análisis por Conglomerados , Diabetes Mellitus Experimental/sangre , Electroforesis en Gel Bidimensional , Hemoglobina Glucada/química , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/metabolismo , Glicoproteínas/química , Glicosilación , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Proteómica , Albúmina Sérica/químicaRESUMEN
Glycation, a non-enzymatic reaction between glucose and protein is the primary cause of diabetic complications. Albumin, the most abundant plasma protein undergoes glycation both in vivo and in vitro. The influence of albumin on glycation of less abundant proteins has not been addressed. For the first time, we show that albumin competitively inhibits the glycation of less abundant proteins. This study suggests that at least in the initial stages of diabetes, albumin may protect other proteins from glycation.